Urolithiasis, or urinary stone disease, is a common urinary tract problem afflicting more than 10% of the U.S. population. Urinary tract stones are most frequently (70%) composed of calcium oxalate alone or calcium oxalate mixed with calcium phosphate. Thus, the management of oxalate in individuals susceptible to urolithiasis is especially important (U.S. Pat. No. 5,286,495).
The majority of oxalate in plasma and urine is derived from the endogenous metabolism of ascorbic acid, glyoxylate, and to a lesser degree, tryptophan. In addition, between 10% and 20% of the urinary oxalate is absorbed from the diet, especially through ingestion of leafy vegetables such as spinach and rhubarb. Ingestion of ethylene glycol, diethylene glycol, xylitol, and excess ascorbic acid can lead to excess levels of oxalate following metabolic conversion. Use of methoxyflurane as an anaesthetic can also lead to oxalosis. Aspergillosis, which is an infection involving an oxalate-producing fungus, can lead to production and deposition of calcium oxalate (U.S. Pat. No. 5,286,495). It is believed that lowering the oxalate levels in the plasma, and subsequently the urine, would decrease the incidence of calcium oxalate stone formation.
Excess serum oxalic acid levels can also be related to genetic disorders. Primary hyperoxaluria is a general term for an irherited disorder which reveals itself in childhood and progresses to renal failure and frequently death in adolescence. It is characterized by high urinary excretion of oxalate and recurring calcium oxalate kidney stones. There are no satisfactory treatments for the two types of primary hyperoxaluria. Hemodialysis and renal transplantation have not been successful in halting the progress of this disease. A controlled diet is also an unsuccessful treatment for primary hyperoxaluria (U.S. Pat. No. 5,286,495).
Oxalate toxicity can also cause the poisoning of livestock, who may graze on oxalate-rich pastures containing plants which are high in oxalic acid such as Halogeton glomeratus, Bassia hyssopifolia, Oxalis pescaprae, and Setaria sphacelata, or grains infected with the oxalate-producing fungi Aspergillus niger. Chronic poisoning is often accompanied by appetite loss and renal impairment. Acute toxicity can lead to tetany, coma and death (Hodgkinson, A. [1977] Oxalic acid in biology and medicine, London: Academic Press, pp 220-222).
Three mechanisms for oxalate catabolism are known: oxidation, decarboxylation, and activation followed by decarboxylation (Hodgkinson, A. [1977], supra at 119-124). Oxalate oxidases are enzymes that are found in mosses, higher plants, and possibly fungi which catalyze the oxidation of oxalate to hydrogen peroxide plus carbon dioxide: (COOH).sub.2 +O.sub.2 .fwdarw.2CO.sub.2 +H.sub.2 O.sub.2. Oxalate decarboxylases are enzymes which produce CO.sub.2 and formate as products of oxalate degradation. An oxalate decarboxylase found in fungi catalyzes the decarboxylation of oxalic acid to yield stoichiometric quantities of formic acid and CO.sub.2 : (COOH).sub.2 .fwdarw.CO.sub.2 +HCOOH. Varieties of both aerobic and anaerobic bacteria can also degrade oxalic acid. An activation and decarboxylation mechanism is used for degradation of oxalate in Pseudomonas oxalaticus and other bacteria (U.S. Pat. No. 5,286,495).
Oxalobacter fonnigenes is a gram-negative anaerobe found in the soil and also in the mammalian intestine, where it plays a significant role in degradation of dietary oxalic acid. A critical limitation of anaerobic growth for this and other anaerobes is explained by the "uncoupling model". During fermentative growth, fermentation end products which include organic acids and alcohols build up in the cytoplasm leading to an acidification of the cytoplasm and a reduction in the internal pH to critical levels. The organic acids act as protonophores; a build up of which results in an inward flux of H.sup.+. The rapid influx of H.sup.+ counteracts the natural proton extrusion mechanisms needed to alkalinize the cytoplasm. The net result is a breakdown of the proton motive force essential to energy requiring membrane associated processes such as active transport of solutes and ions. Also associated with this phenomenon is a diminishing of intracellular ATP generation which has been observed to decrease growth yield. Kashket, E. R. (1987) FEMS Microbiol. Rev. 46:233-244. Large scale fermentation is frequently utilized by those in the biotechnical and pharmaceutical industries for product production. It would therefor be beneficial for industrial processes to have a system which is capable of ameliorating the energy deficit produced during fermentative growth and thereby increasing culture longevity and product yield.
Oxalobacter formigenes derives metabolic energy from the decarboxylation of oxalate during a "proton-motive metabolic cycle". See Anantharam et al. (1989) J. Biol. Chem. 264, 7244-7250 and Maloney, P. C. (1995) Curr. Opin. Cell Biol. 6:571-582. In this cycle, the entry of divalent oxalate is coupled to the exit of its decarboxylation product, monovalent formate, resulting in formation of a membrane potential that is internally negative. Because the intracellular oxalate decarboxylation consumes a cytosolic proton, the entry of negative charge is accompanied in a stoichiometric fashion by appearance of an internal hydroxyl anion. As a result, the combined activity of the vectorial antiport reaction and the scalar decarboxylation step comprises a thermodynamic proton pump. In this way, O. formigenes establishes the proton-motive force required for both the synthesis of ATP, by reversal of a DCCD (dicyclohexylcarbodiimide)-sensitive ATPase, and for the support of other membrane reactions requiring a proton-motive force (See FIG. 1).
Experiments based on the reconstitution of activity from crude detergent extracts suggest the oxalate/formate exchange reaction is mediated by a conventional membrane carrier. See Anantharam et al. (1989) J. Biol. Chem. 264:7244-7250. This reasoning is reinforced by the finding that oxalate transport is catalyzed by a single protein, OxlT, whose SDS-PAGE mobility (ca. 38 kDa) resembles that of other bacterial carrier protein. See Ruan et al. (1992) J. Biol. Chem. 267:10537-10543. For this reason, the present invention includes the cloning and sequencing of OxlT.
Although OxlT is the sole protein required for import of oxalate and export of formate, two other proteins are required for the decarboxylation of oxalate in O. formigenes: oxalyl-CoA decarboxylase (Lung et al. (1991) Amer. J. Kidney Dis. 17:381-385) and formyl-CoA transferase (Sidhu et al. (1997) J. Bacteriol. 179:3378-3381). The isolation of the three proteins will allow the reconstitution of the thermodynamic proton pump from Oxalobacter formigenes. By reconstituting expression of the genes encoding the three proteins in a fermentative bacterium, cell survival could be prolonged during production of a cell product by using oxalate as an additional energy source resulting in a intracellular increase in ATP production. In this manner the invention could be used in industrial fermentation processes to increase product yield by extending culture longevity which is adversely affected by low intracellular ATP levels or by low proton motive force values. It has been demonstrated that individuals with enteric hyperoxauria and recurrent calcium oxalate stone formation lack intestinal colonization by Oxalobacter formigenes. See Allison et al. (1986) J. Nutr. 116:455-460; Goldkin et al. (1985) Am. J. Gastroenterol. 80:860; and Kleinschmidt et al. (1993) Urolithiasis 2. Plenum Press, New York, N.Y. It can therefore be envisioned that the reconstituted pump may have medical and veterinary applications in the reduction of elevated serum oxalate levels associated with the formation of kidney stones and other medical disorders.